Mutations in the mitochondrial (mt) genome are correlated with cancer and aging. G-quadruplexes (G4), dynamic DNA structures that arise in guanine (G)-rich templates, potentially stall the mt replisome and thereby promote mutagenesis. We used computational analyses of genome sequence data from two Italian cohorts to demonstrate an association between G4s and mt variation. Using the software G4Hunter to predict G4-forming regions in mtDNA, we found statistically significant enrichment of mutations in stable G4 regions. In vitro biochemical data demonstrated that G4s potently block the mt replicative polymerase gamma (Pol ). Addition of mt replisome-associated factors including TWINKLE helicase and mt single-strand binding protein were unable to stimulate pol synthesis through the G4 block. We showed that other mt polymerases further catalyzes error-prone nucleotide incorporation into G4 structures, suggesting its involvement in G4 bypass with accompanying increased risk of mutation. Altogether, the computational and biochemical approaches indicate that mt point mutations are enriched at stable G4 structures, consistent with replisome stalling at G-quadruplexes and reliance on error-prone DNA synthesis.